Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example.

Phosphorylation events catalyzed by protein kinases represent one of the most prevalent as well as important regulatory posttranslational modifications, and dysregulation of protein kinases is associated with the pathogenesis of different diseases. Therefore, interest in developing potent small molecule kinase inhibitors has increased enormously within the last two decades. A critical step in the development of new inhibitors is cell-free in vitro testing with the intention to determine comparable parameters like the commonly used IC50 value. However, values described in the literature are often biased as experimental setups used for determination of kinase activity lack comparability due to different readout parameters, insufficient normalization or the sheer number of experimental approaches. Here, we would like to hold a brief for highly sensitive, radioactive-based in vitro kinase assays especially suitable for kinases exhibiting autophosphorylation activity. Therefore, we demonstrate a systematic workflow for complementing and validating results from high-throughput screening as well as increasing the comparability of enzyme-specific inhibitor parameters for radiometric as well as non-radiometric assays. Using members of the CK1 family of serine/threonine-specific protein kinases and established CK1-specific inhibitors as examples, we clearly demonstrate the power of our proposed workflow, which has the potential to support the generation of more comparable data for biological characterization of kinase inhibitors.

Influence of obesity on remodeling of lung tissue and organization of extracellular matrix after blunt thorax trauma.

BACKGROUND: Previously, it has been shown that obesity is a risk factor for recovery, regeneration, and tissue repair after blunt trauma and can affect the rate of muscle recovery and collagen deposition after trauma. To date, lung tissue regeneration and extracellular matrix regulation in obese mice after injury has not been investigated in detail yet. METHODS: This study uses an established blunt thorax trauma model to analyze morphological changes and alterations on gene and protein level in lean or obese (diet-induced obesity for 16 ± 1 week) male C57BL/6 J mice at various time-points after trauma induction (1 h, 6 h, 24 h, 72 h and 192 h). RESULTS: Morphological analysis after injury showed lung parenchyma damage at early time-points in both lean and obese mice. At later time-points a better regenerative capacity of lean mice was observed, since obese animals still exhibited alveoli collapse, wall thickness as well as remaining filled alveoli structures. Although lean mice showed significantly increased collagen and fibronectin gene levels, analysis of collagen deposition showed no difference based on colorimetric quantification of collagen and visual assessment of Sirius red staining. When investigating the organization of the ECM on gene level, a decreased response of obese mice after trauma regarding extracellular matrix composition and organization was detectable. Differences in the lung tissue between the diets regarding early responding MMPs (MMP8/9) and late responding MMPs (MMP2) could be observed on gene and protein level. Obese mice show differences in regulation of extracellular matrix components compared to normal weight mice, which results in a decreased total MMP activity in obese animals during the whole regeneration phase. Starting at 6 h post traumatic injury, lean mice show a 50% increase in total MMP activity compared to control animals, while MMP activity in obese mice drops to 50%. CONCLUSIONS: In conclusion, abnormal regulation of the levels of extracellular matrix genes in the lung may contribute to an aberrant regeneration after trauma induction with a delay of repair and pathological changes of the lung tissue in obese mice.

Influence of bariatric surgery on the peripheral blood immune system of female patients with morbid obesity revealed by high-dimensional mass cytometry.

Introduction: Obesity is associated with low-grade chronic inflammation, altered levels of adipocytokines, and impaired regulation of gastrointestinal hormones. Secreted, these factors exert immunostimulatory functions directly influencing peripheral immune cells. Methods: In the realm of this study, we aimed to investigate the composition and activation status of peripheral blood immune cells in female patients with morbid obesity compared to lean controls using high-dimensional mass cytometry. Besides, we also assessed the influence of bariatric surgery with respect to its ability to reverse obesity-associated alterations within the first-year post-surgery. Results: Patients with morbid obesity showed typical signs of chronic inflammation characterized by increased levels of CRP and fibrinogen. Apart from that, metabolic alterations were characterized by increased levels of leptin and resistin as well as decreased levels of adiponectin and ghrelin compared to the healthy control population. All these however, except for ghrelin levels, rapidly normalized after surgery with regard to control levels. Furthermore, we found an increased population of monocytic CD14+, HLA-DR-, CD11b+, CXCR3+ cells in patients with morbid obesity and an overall reduction of the HLA-DR monocytic expression compared to the control population. Although CD14+, HLA-DR-, CD11b+, CXCR3+ decreased after surgery, HLA-DR expression did not recover within 9 - 11 months post-surgery. Moreover, compared to the control population, patients with morbid obesity showed a perturbed CD4+ T cell compartment, characterized by a strongly elevated CD127+ memory T cell subset and decreased naïve T cells, which was not recovered within 9 - 11 months post-surgery. Although NK cells showed an activated phenotype, they were numerically lower in patients with morbid obesity when compared to healthy controls. The NK cell population further decreased after surgery and did not recover quantitatively within the study period. Conclusions: Our results clearly demonstrate that the rapid adaptions in inflammatory parameters and adipocytokine levels that occur within the first year post-surgery do not translate to the peripheral immune cells. Apart from that, we described highly affected, distinct immune cell subsets, defined as CD127+ memory T cells and monocytic CD14+, HLA-DR, CD11b+, CXCR3+ cells, that might play a significant role in understanding and further decoding the etiopathogenesis of morbid obesity.

Zebrafish as model system for the biological characterization of CK1 inhibitors.

Introduction: The CK1 family is involved in a variety of physiological processes by regulating different signaling pathways, including the Wnt/ß-catenin, the Hedgehog and the p53 signaling pathways. Mutations or dysregulation of kinases in general and of CK1 in particular are known to promote the development of cancer, neurodegenerative diseases and inflammation. There is increasing evidence that CK1 isoform specific small molecule inhibitors, including CK1δ- and CK1ε-specific inhibitors of Wnt production (IWP)-based small molecules with structural similarity to benzimidazole compounds, have promising therapeutic potential. Methods: In this study, we investigated the suitability of the zebrafish model system for the evaluation of such CK1 inhibitors. To this end, the kinetic parameters of human CK1 isoforms were compared with those of zebrafish orthologues. Furthermore, the effects of selective CK1δ inhibition during zebrafish embryonic development were analyzed in vivo. Results: The results revealed that zebrafish CK1δA and CK1δB were inhibited as effectively as human CK1δ by compounds G2-2 with IC50 values of 345 and 270 nM for CK1δA and CK1δB versus 503 nM for human CK1δ and G2-3 exhibiting IC50 values of 514 and 561 nM for zebrafish CK1δA and B, and 562 nM for human CK1δ. Furthermore, the effects of selective CK1δ inhibition on zebrafish embryonic development in vivo revealed phenotypic abnormalities indicative of downregulation of CK1δ. Treatment of zebrafish embryos with selected inhibitors resulted in marked phenotypic changes including blood stasis, heart failure, and tail malformations. Conclusion: The results suggest that the zebrafish is a suitable in vivo assay model system for initial studies of the biological relevance of CK1δ inhibition.

Recent Developments in Mouse Trauma Research Models: A Mini-Review.

The urgency to investigate trauma in a controlled and reproducible environment rises since multiple trauma still account for the most deaths for people under the age of 45. The most common multiple trauma include head as well as blunt thorax trauma along with fractures. However, these trauma remain difficult to treat, partially because the molecular mechanisms that trigger the immediate immune response are not fully elucidated. To illuminate these mechanisms, investigators have used animal models, primarily mice as research subjects. This mini review aims to 1) emphasize the importance of the development of clinically relevant murine trauma research, 2) highlight and discuss the existing conflict between simulating clinically relevant situations and elucidating molecular mechanisms, 3) describe the advantages and disadvantages of established mouse trauma models developed to simulate clinically relevant situations, 4) summarize and list established mouse models in the field of trauma research developed to simulate clinically relevant situations.

CK1 Is a Druggable Regulator of Microtubule Dynamics and Microtubule-Associated Processes.

Protein kinases of the Casein Kinase 1 family play a vital role in the regulation of numerous cellular processes. Apart from functions associated with regulation of proliferation, differentiation, or apoptosis, localization of several Casein Kinase 1 isoforms to the centrosome and microtubule asters also implicates regulatory functions in microtubule dynamic processes. Being localized to the spindle apparatus during mitosis Casein Kinase 1 directly modulates microtubule dynamics by phosphorylation of tubulin isoforms. Additionally, site-specific phosphorylation of microtubule-associated proteins can be related to the maintenance of genomic stability but also microtubule stabilization/destabilization, e.g., by hyper-phosphorylation of microtubule-associated protein 1A and RITA1. Consequently, approaches interfering with Casein Kinase 1-mediated microtubule-specific functions might be exploited as therapeutic strategies for the treatment of cancer. Currently pursued strategies include the development of Casein Kinase 1 isoform-specific small molecule inhibitors and therapeutically useful peptides specifically inhibiting kinase-substrate interactions.

Phenotypic Discovery of Triazolo[1,5-c]quinazolines as a First-In-Class Bone Morphogenetic Protein Amplifier Chemotype.

Phenotypic drug discovery (PDD) continues to fuel the research and development pipelines with first-in-class therapeutic modalities, but success rates critically depend on the quality of the underlying model system. Here, we employed a stem cell-based approach for the target-agnostic, yet pathway-centric discovery of small-molecule cytokine signaling activators to act as morphogens during development and regeneration. Unbiased screening identified triazolo[1,5-c]quinazolines as a new-in-class in vitro and in vivo active amplifier of the bone morphogenetic protein (BMP) pathway. Cellular BMP outputs were stimulated via enhanced and sustained availability of BMP-Smad proteins, strictly dependent on a minimal BMP input. Holistic target deconvolution unveiled a unique mechanism of dual targeting of casein kinase 1 and phosphatidyl inositol 3-kinase isoforms as key effectors for efficient amplification of osteogenic BMP signaling. This work underscores the asset of PDD to discover unrecognized polypharmacology signatures, in this case significantly expanding the chemical and druggable space of BMP modulators.

Regulation of MHC I Molecules in Glioblastoma Cells and the Sensitizing of NK Cells.

Immunotherapy has been established as an important area in the therapy of malignant diseases. Immunogenicity sufficient for immune recognition and subsequent elimination can be bypassed by tumors through altered and/or reduced expression levels of major histocompatibility complex class I (MHC I) molecules. Natural killer (NK) cells can eliminate tumor cells in a MHC I antigen presentation-independent manner by an array of activating and inhibitory receptors, which are promising candidates for immunotherapy. Here we summarize the latest findings in recognizing and regulating MHC I molecules that affect NK cell surveillance of glioblastoma cells.

Obesity Prolongs the Inflammatory Response in Mice After Severe Trauma and Attenuates the Splenic Response to the Inflammatory Reflex.

Thoracic traumas with extra-thoracic injuries result in an immediate, complex host response. The immune response requires tight regulation and can be influenced by additional risk factors such as obesity, which is considered a state of chronic inflammation. Utilizing high-dimensional mass and regular flow cytometry, we define key signatures of obesity-related alterations of the immune system during the response to the trauma. In this context, we report a modification in important components of the splenic response to the inflammatory reflex in obese mice. Furthermore, during the response to trauma, obese mice exhibit a prolonged increase of neutrophils and an early accumulation of inflammation associated CCR2+CD62L+Ly6Chi monocytes in the blood, contributing to a persistent inflammatory phase. Moreover, these mice exhibit differences in migration patterns of monocytes to the traumatized lung, resulting in decreased numbers of regenerative macrophages and an impaired M1/M2 switch in traumatized lungs. The findings presented in this study reveal an attenuation of the inflammatory reflex in obese mice, as well as a disturbance of the monocytic compartment contributing to a prolonged inflammation phase resulting in fewer phenotypically regenerative macrophages in the lung of obese mice.

Influence of Obesity on the Organization of the Extracellular Matrix and Satellite Cell Functions After Combined Muscle and Thorax Trauma in C57BL/6J Mice.

Obesity has been described as a major factor of health risk in modern society. Next to intricately linked comorbidities like coronary artery disease or diabetes, an influence of obesity on regeneration after muscle injury has been described previously. However, the influence of obesity on tissue regeneration in a combined trauma, merging the more systemic influence of a blunt lung trauma and the local blunt muscle trauma, has not been investigated yet. Therefore, the aim of this study was to investigate the influence of obesity on regeneration in a mouse model that combined both muscle and thorax trauma. Using gene expression analysis, a focus was put on the structure as well as the organization of the extracellular matrix and on functional satellite cell physiology. An increased amount of debris in the lung of obese mice compared to normal weight mice up to 192 h after combined trauma based on visual assessment can be reported which is accompanied by a decreased response of Mmp2 in obese mice. Additionally, a delayed and elongated response of inhibitor genes like Timp1 has been revealed in obese mice. This elongated response to the trauma in obese mice can also be seen in plasma based on increased levels of pro-inflammatory chemo- and cytokines (IL-6, MCP-1, and IL 23) 192 h post trauma. In addition to changes in the lung, morphological analysis of the injured extensor iliotibialis anticus of the left hind leg in lean and diet-induced obese mice revealed deposition of fat in the regenerating muscle in obese animals hindering the structure of a compact muscle. Additionally, decreased activation of satellite cells and changes in organization and build-up of the ECM could be detected, finally leading to a decreased stability of the regenerated muscle in obese mice. Both factors contribute to an attenuated response to the trauma by obese mice which is reflected by a statistically significant decrease in muscle force of obese mice compared to lean mice 192 h post trauma induction.

Development of a simplified purification method for a novel formaldehyde dismutase variant from Pseudomonas putida J3.

Formaldehyde dismutase (FDM) is a very interesting enzyme, due to the fact that it comprises an internal cofactor regeneration mechanism. The FDM, therefore, is able to catalyze redox reactions independent of exogenous cofactor addition, rendering the enzyme powerful for industrial applications. Currently, only one enzyme of this type has been characterized enzymatically. Furthermore, only one additional DNA-sequence with high homology to FDM has been published. In this work, we identified a new variant of a formaldehyde dismutase gene (fdm) in the Pseudomonas putida J3 strain. To isolate and characterize the enzyme, we developed a simplified method for its purification. This purification is based on a C-terminal 6xHis-tag, which enables functional expression of the enzyme in E. coli and a one-step purification method. In addition, we tested several expression systems for optimal yields and combined this with co-expression of the chaperonins GroESL. Using this simplified and rapid method, we are now able to produce sufficient material in reproducible quality and quantity for application tests with the enzyme. The newly identified enzyme will be applied in a redox cascade for biomethanol production from biogas and shows potential for further industrial biotransformation with integrated cofactor recycling.